食源性沙门氏菌磁性纳米粒子RT-PCR检测方法的研究

建立适合食源性沙门氏菌的RT-PCR 检测体系。根据沙门氏菌的转录起始因子rpoD 设计引物Salmrpod2/5,以rpoD 基因的mRNA 为检测对象,在样品制备时采用新型的磁性纳米粒子分离mRNA 技术,建立快速检测食品中沙门氏菌的逆转录聚合酶链式反应(RT-PCR)的方法。结果表明,Salmrpod2/5 引物能有效地将沙门氏菌与其他亲缘关系较近的肠杆菌科细菌区分开来。样品中沙门氏菌添加实验表明,该方法对沙门氏菌的检测下限为10CFU/25ml,检测时间少于18h。该方法具有灵敏、特异、快速的特点,适宜于在食品卫生行业中进行推广及应用。     

鱼腥草干燥和贮藏过程中化学成分含量及抑菌效果的测定

以鱼腥草地上部分和地下部分为材料,研究了在室温干燥和贮藏条件下,鱼腥草化学成分含量和抑菌效果的变化。结果表明:鱼腥草在干燥过程中VC、脂肪、黄酮呈下降趋势,总糖含量上升,蛋白质含量相对稳定;在贮藏过程中VC、脂肪、蛋白质、黄酮含量随时间的延长而降低,而总糖含量逐渐增加。鱼腥草干燥0d和1d后,样品挥发油对金黄色葡萄球菌和八叠球菌的最小抑菌浓度(MIC)均为1/2×10-3ml/ml;干燥2、4、8d后,挥发油对两种细菌的MIC为10-3ml/ml;干燥16d后,挥发油对两种细菌的MIC上升到2×10-3ml/ml;贮藏过程中随着时间的延长MIC逐渐增大。     

基于近红外光谱法检测棉/聚酯/氨纶织物的纤维含量

应用近红外光谱法对三组分棉/聚酯/氨纶混纺织物进行纤维定量分析。以200个棉/聚酯/氨纶样品为校正集样品,利用偏最小二乘法建立棉/聚酯/氨纶校正模型。用100个棉/聚酯/氨纶样品作为验证,并对20个样品进行预测。对预测结果进行方差分析,与传统方法 GB/T 2910的结果不存在显著差异,模型的各项指标均达到实际检测的要求,缩短了检测时间,提高了检测效率。该方法不需要化学试剂,精度高,且环保。     

瓶装天然矿泉水饮料的微生物：国家标准和微生物超标问题

对在市场采集的33份瓶装天然矿泉水进行了微生物卫生指标监测,发现细菌菌落总数最高含量为3.8×10~4CFU/ml,最低<10CFU/ml,平均390CFU/ml,按国家饮料的菌落总数标准有76%的样品不合格;大肠菌群有9%的样品不合格,33份样品致病菌指标全部合格,发现不同季节样品的菌落总数含量有所不同;对不同年份的样品微生物含量情况作了分析。还对瓶装天然矿泉水的微生物存在特性,卫生学意义及国标的合适性等进行了讨论。     

发酵乳制品中乳酸菌的流式检测方案探索与研究

目的 建立基于流式细胞术的发酵乳制品中乳酸菌快速计数方法, 实现发酵乳制品中乳酸菌的快速定量检测。方法 使用5(6)-羧基荧光素二乙酸酯（5-(and 6)-Carboxyfluorescein diacetate,cFDA） 和碘化丙啶（Propidium iodid,PI）2种荧光染料对乳酸菌进行染色,经流式细胞仪检测乳酸菌的荧光信号,从而实现细菌定量计数。在分析过程中对染色剂浓度、荧光通道阈值进行优化,并将流式计数结果与现行国标方法进行对比研究。结果 在样品浓度为104 CFU/mL(g)时,通过0.05 μg/mL的PI和10 μmol/L的cFDA染色,在绿色荧光通道增益为100,红色荧光通道增益达到1000时,能够达到最佳的流式检测条件,且样品检测结果与现行国标检测方法存在显著的正相关关系（P<0.01）,2种方法测试结果相关系数值为0.949。结论 流式细胞术具有灵敏度高、检测时间短、稳定性高、重现性好等优点,对于货架期普遍较短,放行压力大的乳制品生产企业来说,能够缩短检测时间,实现产品快速放行上架。     

上转磷光免疫层析检测肠出血性大肠杆菌O157

目的建立一种肠出血性大肠杆菌O157的上转磷光免疫层析快速检测方法。方法利用上转换磷光标记和双抗体夹心免疫层析技术检测大肠杆菌O157,评价了该法的敏感性和特异性,并用于模拟污染食品样品的检测。结果该法能在40min内完成检测,应用于不同的肠杆菌科细菌如其他大肠杆菌、沙门菌、志贺菌、变形杆菌、产气肠杆菌、枸橼酸杆菌、沙雷菌、耶尔森菌以及葡萄球菌、副溶血弧菌、单增李斯特菌等23种28株常见细菌,未发现有交叉反应,检测灵敏度为5×103CFU/ml,每次最低可检测500个细菌,对奶粉、咖啡粉、饼干、蛋糕、绿豆糕、果冻、燕窝、果汁等样品中的人工染菌均可检测,最低检测浓度为5×103CFU/ml。结论肠出血性大肠杆菌O157上转磷光免疫层析方法简便快速,特异性和灵敏性好,适用于现场的快速检测。     

冰核活性细菌Xanthomonas ampelinaTS206实验室水平发酵生产工艺条件优化的研究

优化冰核活性细菌XanthomonasampelinaTS206实验室水平发酵生产的工艺条件为将冰核活性细菌应用在工业化生产提供了重要的技术资料。本文对冰核活性细菌XanthomonasampelinaTS206实验室水平的发酵生产工艺条件进行了系统的研究,结果表明这种冰核活性细菌的最适培养条件为:培养温度18℃,装液量40ml/250ml,摇床转速180r/min,接种量3%,发酵液的初始pH8。实验还确定了冰核活性细菌在发酵48h时达到对数生长期。采用上述发酵条件培养XanthomonasampelinaTS206可以使细菌在冰核活性不降低的条件下发酵液的菌体浓度达到2.87×1010个/ml,菌体干重增加了37.7%。     

速冻水饺细菌总数FISH检测技术的优化

探索荧光原位杂交(FISH)技术检测速冻水饺中细菌总数的可行性及特异性,优化样品的快速、准确和便捷的检测体系。采用TAMRA标记的EUB338探针与大肠杆菌进行FISH实验条件的优化,并利用优化的FISH技术对4种速冻水饺中的8个样品进行检测和计数。应用FISH技术检测速冻水饺中细菌总数,理想的样品预处理实验条件为:热固定2 h,4%多聚甲醛固定20 min,系列乙醇脱水5 min,杂交条件为杂交温度为46℃,杂交液中去离子甲酰胺浓度为20%,杂交时间3h,洗脱液中NaCl浓度为225 mmol/L;检测的8个样品中,有2例细菌总数超标。FISH可以特异性的检出速冻水饺中的细菌总数,简便,检测周期短,灵敏度高,为保障食品安全提供更多的理论支持。     

莴苣中两种致泻性大肠杆菌的富集及DNA提取方法的优化

本研究对人工接种肠出血性大肠杆菌O157∶H7和肠侵袭性大肠杆菌的莴苣样品中菌体的富集和DNA提取方法进行了优化。研究中比较了不同过滤膜组合对菌体的富集效果,筛选了莴苣样品中细菌DNA提取的最适方法,建立了多重PCR方法检测人工污染莴苣样品。结果表明,采用尼龙膜15μm+混合膜0.22μm的组合富集细菌、试剂盒法提取样品中细菌DNA,多重PCR检测的检出限降低10倍,检测时间节省1.5 h。本研究可快速、简便、灵敏的应用于莴苣检测中,具有很高的实际应用价值。      

沙拉沙星体外诱导鼠伤寒沙门菌耐药实验

目的为获得体外鼠伤寒沙门菌(Salmonella typhimurium)对盐酸沙拉沙星(sarafloxacin hydrochloride)的活性耐药菌株,研究诱导其产生耐药的盐酸沙拉沙星最低浓度,为评价食品中沙拉沙星残留导致的微生物耐药提供依据。方法设置盐酸沙拉沙星0.001、0.002 5、0.005、0.025、0.05、0.1μg/ml实验组和空白对照组、NaOH溶剂对照组,采用NCCLS法测试最小抑菌浓度,对鼠伤寒沙门菌进行耐药诱导,耐药判断为≥8×MIC(0.25μg/ml)。对产生耐药的鼠伤寒沙门菌的gyrA基因片段进行PCR扩增,焦磷酸凝胶测序法鉴定gyrA基因常见5个位点观察突变。结果盐酸沙拉沙星在0.005μg/ml水平传到第10代时,MIC增大32倍,抑菌浓度增加到1μg/ml,耐药菌增殖停止;传到第25代时,鼠伤寒沙门菌gyrA基因Ser83位点发生突变,获得多株稳定、有生物活性的耐药菌株。沙拉沙星浓度≥0.025μg/ml时,细菌不生长;沙拉沙星浓度≤0.002 5μg/ml时细菌生长,不产生耐药。结论沙拉沙星浓度为0.005μg/ml时可体外诱导鼠伤寒沙门菌产生耐药,经传代后获得具生物活性的耐药菌株。     

实时光电微生物法快速检测生鲜乳中的嗜冷菌

对50份生鲜牛乳样品中的嗜冷菌同时采用实时光电微生物快速检测方法和平板计数法进行检测,分析比较两种检测方法所得的嗜冷菌检测数据。结果表明:采用嗜冷菌检测标准曲线:Lg CFU/mL=-0.417*DT+7.91的实时光电微生物法检测的嗜冷菌含量在2.3~3.7×106CFU/mL,检出时间在3.2~18.1 h。采用平板计数法的检出结果范围在4.0~3.0×106CFU/mL。当实时光电微生物检测方法的检出时间大于18 h时,样品检测结果均为未检出,此时平板计数法的检测结果均为<1 CFU/mL。实时光电微生物法检测结果对数值与平板计数法检测结果对数值的差值范围在-0.9~0.8,两者差值的绝对值均<1。采用实时光电微生物法和平板计数法两种检测方法的检测结果相当吻合,实时光电微生物法检测生鲜牛乳中嗜冷菌具有良好的适用性,可大大缩短检测时间,且方法准确、简便易行。     

食品中单核细胞增生李斯特氏菌两种定量检测方法的比较

研究比较不同杂菌污染条件下平板计数(Plate counting)法和稀释培养计数(Most probable number counting)法检测食品中单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)含量的准确性,并探讨冷藏和冷冻食品,MPN保存过程中LM的数量增长情况。采用国标法中的平板计数法和MPN计数法对不同程度人工污染杂菌的牛奶、凉拌菜和盐水鸭分别进行LM含量的检测,并用MPN计数法对冷藏(2~8℃)和冷冻(-20℃)条件下保存的牛奶、凉拌菜和盐水鸭进行LM含量的检测。结果表明,杂菌染菌浓度较低时(LM含量与杂菌含量比为10:1), LM检出限较高(≥ 100 CFU/g (mL)),平板计数法检测LM含量的准确性较高,而杂菌染菌的初始浓度较高时(LM含量与杂菌含量比为1:10),LM检出限较低(< 100 CFU/g (mL)),MPN计数法检测LM含量的准确性较高;牛奶、凉拌菜和盐水鸭在经过不同冷藏和冷冻保存时间后LM数量对比差异有统计学意义(p<0.05),且食品中LM数量随着冷藏和冷冻保存时间的增加而增多。     

应用流式细胞技术快速定量检测UHT奶产品中的微生物

采用基于单个活细胞的新型荧光标记技术,精确区分UHT奶样品中的活菌细胞与死细胞以及其他大颗粒物质,并应用流式细胞技术(flow cytometry,FCM)对UHT奶产品进行微生物快速定量检测。通过与传统的平板计数检测方法进行比对,结果表明,FCM方法的检测范围为101~107CFU/mL,远远高于平板计数法。2种方法的计数结果相关性分析表明,在一定菌液浓度范围内,FCM方法的定量结果与平板计数法线性相关良好,且对不同类型菌种都能精确标记定量,是更为快速、准确的检测方法。     

Determination of somatic cells in milk by solid phase cytometry

The somatic cell count of milk is routinely determined by the fluoro-opto-electron method and sometimes by the direct epifluorescent filter technique (DEFT). This paper investigates the potential of solid phase cytometry (SPC), a novel technique combining aspects of both the fluoro-opto-electronic method and epifluorescence microscopy for somatic cell counting. In SPC, cells are retained on a membrane filter, fluorescently labelled and automatically detected on the entire membrane filter by means of a laser scanning instrument ChemScan). Fluorescent spots can be visually inspected by an epifluorescence microscope with a computer-driven moving stage. The performance of SPC was compared with that of the fluoro-opto-electronic method using a Fossomatic 360 instrument for 68 milk samples with varying somatic cell counts (10(3)-10(6)/ml). The sample throughput and repeatability of SPC were inferior to those of' the Fossomatic method and statistical analysis of the method comparison data using the approach of J. M. Bland & D. G. Altman (The Lancet 1986 February 8 pp 307-310) revealed a poor comparability between the two methods. Moreover, problems of milk filterability and the interference of fluorescent particles presently hamper the routine application of SPC. Nevertheless, this method represents the first example of the application of SPC to milk.     

Outer membrane protein A (OmpA) may be used as a novel target to enrich and detect Escherichia coli in milk samples

In recent years, food safety incidents caused by Escherichia coli have occurred and have endangered human health. Due to the complex matrix of milk samples and the long pretreatment time, the existing methods cannot quickly detect E. coli in milk samples. It is necessary to enrich the E. coli in the complex matrix to improve the detection sensitivity. The E. coli outer membrane protein A (OmpA) is widely present on the cell membrane of E. coli and may be used as a new target to enrich E. coli. In this study, the purified recombinant OmpA protein was used to immunize BALB/c mice to produce polyclonal antibody. Immunomagnetic beads were combined with the polyclonal antibody to enrich the E. coli in the artificially contaminated milk samples. The products of immunoprecipitation were further used for PCR assay. The bacteria in the PCR sample can be pre-enriched, and the limit of detection is 10 × 10⁰ cfu/mL, which is about 100 times more sensitive than samples not processed by this method. Then, the artificially contaminated milk, coffee, juice, and soybean milk samples were tested separately, and it was found that the E. coli gene could be amplified. The whole analysis time was about 120 min, including the enrichment of bacteria and the detection of eluate. We found that OmpA combined with immunomagnetic beads was more efficient, fast, and convenient than the conventional method. Bacteria can be enriched more efficiently without extracting genomic DNA and culturing bacteria. Therefore, this method has potential value for improving the detection sensitivity and shortening the detection time of E. coli in food samples.     

乳酸菌饮料中乳酸菌微生物学检验技术的分析与探讨

只有活性乳酸菌群≥10^6个／mL的乳酸菌饮料，才能真正起到增强机体免疫功能、延缓机体衰老等保健作用，检测活性乳酸菌的含量成为判断产品质量好坏的重要手段。该文通过大量试验，对国标中乳酸菌检验方法中菌落特征及菌体形态的文字描述作了进一步细化、具体化的实物图像补充，从抽象的文字描述转变成更清晰、形象的图像说明，为准确计数乳酸菌总数提供了依据。     

氢化物—原子荧光法测定乳粉中的微量硒

采用氢化物原子荧光光度法测定乳粉中的硒含量。样品经ＴＣ－Ⅱ型快速消化器用硝酸高氯酸混合酸消化后，直接上机测定，取得了较满意的结果。本方法具有操作简单、分析速度快、灵敏度高的特点。其最低检出限为０．５０ｎｇ／ｍｌ，回收率９０．２０％￣１０７．５％，线性范围为１．０￣２５０．０ｎｇ／ｍｌ，相当系数≥０．９９９７。     

Combined PCR and slot blot assay for detection of Salmonella and Listeria monocytogenes

Detection of Salmonella typhimurium and Listeria monocytogenes by the polymerase chain reaction (PCR) assay coupled with slot blot detection was investigated in this study. After being extracted from diluted bacterial culture with the extraction buffer, bacterial DNA was subjected to PCR. The slot blot assay was optimized and used to detect PCR products. The lowest detection level of this method was 10(3) cfu/ml in the original culture media for both pathogens, or 5 bacterial cells in the PCR reaction. Combined with immunomagnetic separation (IMS) to separate and concentrate bacteria from samples, the detection limit could be 40 cfu/ml of bacteria from milk samples. The whole detection procedure was completed within 7 h. After multiplex PCR (amplification of DNA from two different bacteria in the same PCR tube) and slot blot, a detection level of 10(3) cfu/ml was achieved in the simultaneous detection for both pathogens, which was similar to that of individual detection for each pathogen. The combination of PCR and slot-blot seems to be highly sensitive and time-efficient, and is therefore promising for routine use in the detection of Salmonella and L. monocytogenes in food samples such as milk.     

Capture immunoassay for the diagnosis of bovine mastitis using a monoclonal antibody to polymorphonuclear granulocytes.

A direct capture enzyme-linked immunosorbent assay (ELISA) was developed to measure elevated polymorphonuclear granulocyte (PMN) antigens using horseradish peroxidase (EC 1.11.1.7) conjugated rabbit polyclonal anti-PMN antisera and a monoclonal antibody specific for PMN cells. Optical densities obtained in the ELISA were used to predict the cell counts of milk samples. Predicted counts were not significantly different from actual somatic cell counts (SCC). In a total of 156 bovine milk samples the correlation coefficient between somatic cell counting, taking greater than 500,000 cells/ml as being indicative of mastitis, and the assay was 0.94, yielding an assay sensitivity of 95.2% and a specificity of 97.3%. In further trials the ELISA could detect elevated PMN antigens in milk with SCC as low as 100,000 cells/ml. The results indicate that the monoclonal antibody based direct ELISA has excellent potential in the detection and determination of bovine mastitis.     

PCR 检测乳品中大肠杆菌的研究

用大肠杆菌人工污染乳样品直接或8h 增菌后提取DNA 模板,以大肠杆菌丙氨酸消旋酶基因alr 保守区设计引物,经过PCR 扩增凝胶电泳检测,不增菌条件下全脂乳和脱脂乳的检出限为104CFU/ml,增菌后检出限为1CFU/ml。与国标鉴别培养法比较,PCR 法时间缩短、灵敏度和准确度提高,该法更适宜乳品生产经营的快速实时检测。